Apparatus for chemiluminescent assay and detection

ABSTRACT

An apparatus includes a system for guiding chemiluminescence and a system for preventing a variation in dark currents. The apparatus includes a first light shielding BOX having a sample container holder and a shutter unit therein, the shutter unit including a top plate which is partly formed by a movement of a plate member, and a second light shielding BOX having a photodetector therein. While a measurement is not implemented, the shutter unit is closed to block entrance of stray light to the photodetector, and while a measurement is implemented, the plate member is moved to open the shutter unit, and the tip of the photodetector is inserted into a through hole formed in the top plate, so that the distance between the bottom of the sample container and a sensitive area of the photodetector is reduced to several millimeters or less.

This application is a divisional application of U.S. application Ser.No. 12/034,880 filed on Feb. 21, 2008, now allowed. The entirety ofwhich is incorporated herein by reference.

CLAIM OF PRIORITY

The present application claims priority from Japanese application JP2007-112018 filed on Apr. 20, 2007, the content of which is herebyincorporated by reference into this application.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a chemiluminescence measuring apparatusfor detecting chemiluminescence and bioluminescence of a substance whichis contained in a liquid specimen, with high sensitivity and accuracy.The present invention also relates to a microbe count function fordetecting ATP chemiluminescence of microbes to control a contaminationlevel.

2. Background Art

Microbe monitoring for environmental control of medicinal chemicalmanufacturing plants and the like involves counting of airbornemicroorganisms, falling microorganisms, and adherent microorganisms. Themethod for counting is defined by International Organization forStandardization ISO 14698-1, and the cleanliness measured by the methodis expressed by grades. Airborne microorganisms are generally measuredby methods using the gravity-drop of airborne microorganisms or theairborne microorganism sampler which sucks a certain amount of air asdisclosed in JP Patent Publication (Kokai) No. 2002-153259 A. In themethods, usually, microorganisms are collected on agar plates for acertain period of time to culture, and the cleanliness of theenvironment is evaluated by the number of colonies developed after theculture. The agar plates are generally cultured in a temperaturecontrolled incubator for a few days, and the numbers of developedcolonies are visually counted. The numbers of colonies in the agarplates are averaged to obtain the mean number of airbornemicroorganisms. In a manufacturing facility of aseptic medical productsor Cell Processing Center (CPC) for producing cells which include cleanrooms with a high level of cleanliness, among the above describedgrades, a grade A or B should be consistently maintained and controlled.These grades A and B correspond to the number of particulates in the airof 3,530/m³ or less, and the number of microorganisms of 10 CFU (colonyforming unit)/m³ or less.

Meanwhile, for a contamination control of food, river, sewage disposal,and the like, a method is used in which luciferase and luciferin areadded as chemiluminescence reagents to ATP (adenosine triphosphate) inmicrobes to measure the bioluminescence generated from the ATP. Theobtained luminescence intensity is calculated into the number ofmicrobes as disclosed in JP Patent Publication (Kokai) No. 2000-314738 Aand JP Patent Publication (Kokai) No. 7-83831 A (1995) for example, sothat the contamination level can be controlled. According to FIG. 6 ofNippon Nogeikagaku Kaishi Vol. 78, No. 7, pp. 630-635, 2004, thequantification limit of Escherichia coli is about 100 CFU/mL (with areproducibility of 10.2% after N experiments where N=10). At the sametime, according to FIG. 2 included in the instruction of a reagent kitwhich is shown in FIG. 6 of the above non-patent document, thequantification limit of Escherichia coli is about 200 CFU/mL, and thespecimen solution supplied for measuring chemiluminescence is about 0.1mL, which indicates that the quantification limit of Escherichia coli ofthis method is considered to be about 10 to 20 CFU.

ATP chemiluminescence assay can be applied to the measurement ofairborne microorganisms. That is, microbes and dusts are collected ontoan agar which is held in a Petri dish by an airborne microorganismsampler, and after an addition of a development solution, the number ofairborne microorganisms included in the collected sample is calculatedusing ATP chemiluminescence, so that the number of living microbe, themicroorganisms that are alive, is counted.

ATP chemiluminescence assay is conducted using a reagent kit provided bya certain manufacturer in accordance with the measurement procedurewhich is as follows:

(A) an ATP eliminating reagent is dispensed in a sample solution tube soas to eliminate killed microbes and ATP other than living microbes;(B) another ATP eliminating reagent is dispensed in the sample solutiontube so as to extract ATP from the living microbes;(C) a chemiluminescence reagent is dispensed in the sample solutiontube; and(D) move the tube which contains the chemiluminescence reagent and themixed solutions to a black box of an apparatus for measuring the amountof chemiluminescence.

In such an ATP chemiluminescence assay, in order to measure ATPchemiluminescence with high sensitivity and accuracy, the use of adetector with high sensitivity and the achievement of a highconcentration by an optical arrangement of the detector and achemiluminescence reaction field are the important factors. Moreover, alight shielding set up for constraining the entrance of so-called straylight as much as possible is another important factor because theentrance of stray light which comes from the exterior of the apparatusor chemiluminescent substance into the apparatus decreases the accuracyof chemiluminescence measurement.

First, as to the detector with high sensitivity, conventionally,photomultipliers have been used as a photodetector of a microbe countapparatus which has a luminometer for ATP measurement or a luminometerusing ATP chemiluminescence. When a higher sensitivity is needed, asingle photon counting method (photon counting method) for digitallyprocessing the signals from a photomultiplier is used.

Next, as to the optical arrangement, because a chemiluminescenceintensity is decreased inversely by the distance square from achemiluminescence emitting point, it is considered to be effective toplace a specimen container having a chemiluminescence substance thereincloser to a sensitive area. Also, the chemiluminescence from theluminous point is scattered in a sphere, the optical arrangement whichallows an effective collection of the chemiluminescence to a sensitiveare is important. A chemiluminescence collection efficiency is oftendefined using a solid angle, and according to the definition, asensitive area which is closer to a container and larger relative to theluminous area is important to achieve a higher sensitivity. Also,specular members which surround a container holder are effective tocause chemiluminescence to be forcibly reflected at the specularsurfaces to be introduced to the sensitive area.

Finally, to address stray light (to prevent an entrance of stray light),generally, a photodetector and a specimen container are covered with alight-shielding box, that is, the entire apparatus for chemiluminescentassay and detection is completely covered with a shielding body to blockstray light.

However, a microbe count apparatus which uses ATP chemiluminescence hasa solution control section therein for dispensing and collection of asolution in addition to a photodetector, which increases an area of theapparatus to be shielded, and also the material may includes a luminouselement. This makes it difficult to block an entrance of stray light.

Thus, it is effective to partially shield an apparatus forchemiluminescent assay and detection from light, and JP PatentPublication (Kokai) No. 7-83831 A (1995) discloses a case where aluminometer is used to achieve the partial shielding. Generally, anopenable/closable shutter unit is placed in front of a sensitive area ofa photodetector to shield light (hereinafter, referred to as “doublelight shielding type”). The unit prevents an entrance of light to thechemiluminescence detecting means just prior to a sensitive area.Therefore, no stray light hits a light-receiving element, which preventsdegradation and variation in dark currents due to an accumulation oflights caused by the stray light.

SUMMARY OF THE INVENTION

However, in the above described structure of the double light shieldingtype, when a light shielding set up is placed at the tip of a sensitivearea, an openable/closable shutter unit is provided in front of thesensitive area. The shutter unit can be an obstacle which substantiallyincreases the distance between a sample container and the sensitivearea, and also increases the distance from a luminous point. This maycause a problem of a decreased sensitivity.

In addition, the conventional methods described above could not provideda sufficiently high sensitivity or measurement accuracy for detectingATP chemiluminescence on the order of one microbe level which isrequired in manufacturing facilities of aseptic medical products andCPC, and so generally involves a pre-treatment process for culturing toincrease microbes. This causes a problem that the processes becomecomplicated and more than a half day is spent in the series of processesfor obtaining the test result of cleanliness.

The present invention was made in view of the above situation, and thepresent invention provides an apparatus for chemiluminescent assay anddetection with high sensitivity and accuracy which enables a simpleoperation for chemiluminescence measurement.

In order to solve the above described problems, an apparatus forchemiluminescent assay and detection according to the present inventionincludes: a container for storing a specimen; a holder for holding thecontainer; a photodetector which is provided opposite to the container;a plate member which is provided opposite to the photodetector; a platemember driving section which causes the plate member to be movedrelative to the photodetector; and a photodetector position controlsection which moves the photodetector relative to the container. Thephotodetector is provided opposite to the container via the platemember. The photodetector position control section moves thephotodetector so that, when at least a part of the plate member ismoved, an end surface of the photodetector is placed at the sameposition as that of a surface of the plate member which is opposite tothe photodetector or at a position closer to the container than theopposite surface.

Also, an apparatus for chemiluminescent assay and detection according tothe present invention includes: a container for storing a specimen; aholder for holding the container; a light-shielding housing having a topplate in which a through hole is formed so that the holder is placedover the through hole; a photodetector which is provided in thelight-shielding housing in opposition to the bottom of the container viathe top plate of the light-shielding housing; a photodetector positioncontrol section which moves the photodetector relative to the container;at least one nozzle; at least one solution reservoir; at least onepiping tube; at least one feed pump which is connected to the pipingtube; and a nozzle position control section which causes a nozzle tomove into the container. The photodetector position control sectioncontrols the position of the photodetector so that an end surface of thephotodetector is placed substantially at the same position as that of asurface of the top plate of the light-shielding housing opposite to thephotodetector or at a position closer to the container than the oppositesurface. The nozzle position control section controls the nozzle so thatthe nozzle is inserted into the container.

Moreover, an apparatus for chemiluminescent assay and detectionaccording to the present invention further has a function to measure theamount of microbes. That is, the apparatus for chemiluminescent assayand detection includes: a first light-shielding housing having anopen/close door; a container for storing a specimen; a holder forholding the container; a second light-shielding housing which isaccessible through the open/close door, and has a top plate with athrough hole formed therein so that the holder is placed over thethrough hole; a photodetector which is provided in the secondlight-shielding housing in opposition to the bottom of the container viathe top plate of the second light-shielding housing; a photodetectorposition control section which moves the photodetector relative to thecontainer; a dispensing means which has a nozzle, a solution reservoir,a feed pump, and a solution supply path; a fluid dispensing means whichhas a nozzle, a solution reservoir, a feed pump, and a solution supplypath; and a nozzle position control section which causes a nozzle tomove into the container. The apparatus for chemiluminescent assay anddetection is provided with at least three dispensing means and at leastone fluid dispensing means. The photodetector position control sectioncontrols the position of the photodetector so that an end surface of thephotodetector is placed substantially at the same position as that of asurface of the top plate of the second light-shielding housing which isopposite to the photodetector or at a position closer to the containerthan the opposite surface. The nozzle position control section controlsthe nozzle so that the nozzle is inserted into the container. And then,an ATP-eliminating reagent, ATP extracting reagent, and achemiluminescence solution by ATP-derived are introduced into eachnozzle through the tip thereof so that the amount of microbes can bemeasured using a luminescence intensity of ATP in living microbes.

These and other features of the present invention will be apparent fromthe following description of the best embodiments to implement thepresent invention and the accompanying drawings.

According to the present invention, a double light shielding type box,specifically a second light-shielding box, prevents light accumulationdue to stray light while chemiluminescence is not being measured,thereby a variation in background signals which depend on measurementaccuracy is reduced, and while chemiluminescence is being measured,proximity effect induced by a photodetector and the bottom of a samplecontainer enables a quantitative measurement of ATP of a very lowconcentration, thereby for example, a weak light emission of ATPchemiluminescence in one microbe can be measured with high sensitivityand accuracy, and microbes can be counted one by one.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a view showing an outer structure of an apparatus forchemiluminescent assay and detection according to a first embodiment;

FIG. 1B is a view showing a schematic structure of the apparatus forchemiluminescent assay and detection according to the first embodiment;

FIG. 2A is a view (1) showing the operation principle of the apparatusfor chemiluminescent assay and detection according to the firstembodiment;

FIG. 2B is a view (2) showing the operation principle of the apparatusfor chemiluminescent assay and detection according to the firstembodiment;

FIG. 2C is a view (3) showing the operation principle of the apparatusfor chemiluminescent assay and detection according to the firstembodiment;

FIG. 2D is a view (4) showing the operation principle of the apparatusfor chemiluminescent assay and detection according to the firstembodiment;

FIG. 3A is a view showing a structure (1) of a sample container holderwhich is used in the apparatus for chemiluminescent assay and detectionaccording to the first embodiment;

FIG. 3B is a view showing a structure (2) of a sample container holderwhich is used in the apparatus for chemiluminescent assay and detectionaccording to the first embodiment;

FIG. 3C is a view showing a structure (3) of a sample container holderwhich is used in the apparatus for chemiluminescent assay and detectionaccording to the first embodiment;

FIG. 4 is a view showing a schematic structure of an apparatus forchemiluminescent assay and detection having a dispenser according to asecond embodiment;

FIG. 5A is a view (1) showing the operation principle of the apparatusfor chemiluminescent assay and detection having a dispenser according tothe second embodiment;

FIG. 5B is a view (2) showing the operation principle of the apparatusfor chemiluminescent assay and detection having a dispenser according tothe second embodiment;

FIG. 5C is view (3) showing the operation principle of the apparatus forchemiluminescent assay and detection having a dispenser according to thesecond embodiment;

FIG. 6 is a flowchart illustrating a procedure for measuring ATPchemiluminescence using the apparatus for chemiluminescent assay anddetection having a dispenser according to the second embodiment;

FIG. 7A is a graph showing an ATP chemiluminescence curve relative totime which is obtained using an apparatus for chemiluminescent assay anddetection having a dispenser according to the second embodiment;

FIG. 7B is a graph showing a relationship between ATP concentration andluminescence intensity which is obtained using an apparatus forchemiluminescent assay and detection having a dispenser according to thesecond embodiment;

FIG. 8A is a view showing the outline of a chemiluminescence apparatushaving a function for counting microbes according to a third embodiment;

FIG. 8B is a view showing a chemiluminescence apparatus having afunction for counting microbes according to the third embodiment with alight-shielding stage being drawn outside;

FIG. 9 is a view showing a schematic structure of a chemiluminescenceapparatus having a function for counting microbes according to the thirdembodiment;

FIG. 10 is a flowchart illustrating a procedure for measuring the amountof ATP in living microbes using a chemiluminescence apparatus having afunction for counting microbes according to the third embodiment; and

FIG. 11 is a view showing a state in which a sensitive area of aphotodetector is shielded using a light shielding attachment based onthe operation principle of a chemiluminescence apparatus having afunction for counting microbes according to the third embodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Now, embodiments of the present invention will be described below withreference to the accompanying drawings. However, it should be noted thatthe embodiments are illustrated only as examples to implement thepresent invention, and are not to be construed as limiting the presentinvention. The same reference numerals are given to the common elementsthroughout the drawings.

First Embodiment

FIG. 1 is a schematic view showing a structure of an apparatus forchemiluminescent assay and detection according to a first embodiment.FIG. 1A is an outline view showing a system which includes achemiluminescence measuring apparatus 100 and a control device 3 forcontrolling the chemiluminescence measuring apparatus 100. Thechemiluminescence measuring apparatus 100 includes a first lightshielding BOX 1 and an open/close door 2 which is opened/closed toinstall a sample container. The inner structure of the chemiluminescencemeasuring apparatus 100 is shown in FIG. 1B. FIG. 1B is shown as anexploded view for simplicity of illustration.

A sample container 4 is installed in a sample container holder 5. Thesample container holder 5 is placed over a through hole 8 which isformed in a top plate 7 of a second light shielding BOX 6. The samplecontainer holder 5 is configured to be positioned on the top plate 7when placed thereon. For example, a frame may be provided to the topplate 7 to place the sample container holder 5 at a fixed position, or asquare groove may be formed in the top plate 7 to receive the bottom ofthe sample container holder 5 so that the sample container holder 5 canbe fitted in the groove.

As shown in FIG. 1B, the sample container holder 5 is configured to haveoverlapped two cylinders of different diameters, and is through the topand bottom of the sample container holder 5. The structure of the samplecontainer holder 5 will be explained in detail below by way of otherexamples. The sample container 4 is inserted through an opening of theupper cylinder of the smaller diameter, and is fixed using a flange 4 awhich is provided at the top of the container. Thus, the samplecontainer 4 is mounted to the sample container holder 5 in a state ofbeing hung from the holder 5. When a sample container 4 without a flangeis used, an exclusive stopper or the like (not shown) provided to thesample container 4 may be prepared.

The top plate 7 of the second light shielding BOX 6 is formed so thatthe plate member 9 can be inserted therethrough, and the inserted platemember 9 is able to move in the direction of the y-axis through the topplate by a first actuator 10. The through hole 8 functions as anopenable/closable window in accordance with the movement of the platemember 9.

The second light shielding BOX 6 has a photodetector 13 housed therein.The photodetector 13 is able to move in the direction of the z-axisusing a second actuator 12. The sample container 4, the sample containerholder 5, the center of the through hole 8, and the center of anentrance window 16 of the photodetector 13 are coaxially aligned in thedirection of the z-axis. These elements are aligned when the apparatusis assembled. The first actuator 10 and the second actuator 12 may bethose which are controlled by an electrical supply or an air supply.

The photodetector 13 is generally preferably a photomultiplier(Photomultiplier Tube: PMT) in terms of sensitivity. However, in a casewhere the required sensitivity is not so high as that of a PMT and areduced cost of the apparatus is more important, a semiconductor devicesuch as a photodiode may be used. However, only a system using a PMTwill be described herein. In a PMT (photodetector 13), the parts exceptaround the entrance window 16 is covered with a conductive shield, andin the present invention, the shield is grounded to thechemiluminescence measuring apparatus to prevent electrostatic charge onthe photodetector 13.

FIG. 2 is a view illustrating the operation principle of thechemiluminescence measuring apparatus 100 of FIG. 1.

FIG. 2A is a view showing a state just to start a measurement after theopen/close door 2 is opened to insert the sample container 4 having achemiluminescent material sample 14 therein in the sample containerholder 5 and the open/close door 2 is closed. In the process for placingthe sample container 4, the through hole 8 is closed by the plate member9, thereby any stray light which may enter the inside of the secondlight shielding BOX 6 when the first light shielding BOX 1 is opened iscompletely blocked.

A command from the control device 3 to start a measurement causes theplate member 9 to move in the direction of the y-axis, as shown in FIG.2B. This displaces the bottom of the sample container 4 in a positionopposite to a photocathode surface area 15 and the entrance window 16 ofthe photodetector 13.

Next, the second actuator 12 is driven to cause the photodetector 13 toget closer to the bottom of the container 4 (FIG. 2C). The entrancewindow 16 and a part of the photocathode surface area 15 at the tip ofthe photodetector 13 are inserted in the through hole 8 of the top plate7 to a position closer to the bottom of the sample container 4 in thedirection of the z-axis as compared to the distance between the bottomof the sample container 4 and the plate member 9.

In the state shown in FIG. 2C, a high voltage (HV) is applied to thephotodetector 13 to start a chemiluminescence measurement.

Needless to say, since the second light shielding BOX 6 blocks straylight from the outside of the first light shielding BOX 1, the HV may beapplied before the sample container 4 is placed in the sample containerholder 5. However, for an extraordinary situation of the apparatus suchas breakdown of a driving system of the plate member 9, the HV ispreferably turned off while the open/close door 2 of the first lightshielding BOX 1 is in an open state.

FIG. 2D shows a case where another sample container 4 is used, thecontainer 4 having a smaller volume and a shorter length in thedirection of the z-axis than those of FIGS. 2A-2C. The photodetector 13can be positioned using the second actuator 12 as needed, and also canbe moved to a position above the plate member 9 in the direction of thez-axis, which keeps the distance from the luminous point, specificallythe distance from the bottom of the sample container 4 and thephotocathode surface area 15, to be constant even when the size of thesample container 4 is changed.

Alternatively, both of the sample container 4 and the sample containerholder 5 may be moved to get closer to the photodetector 13. However, inthis case, if the number of the sample to be measured is not one, thatis, there is a plurality of sample containers 4 to be serially andautomatically measured, actuators for each sample container 4 should beprovided to individually control the distances between the samplecontainers 4 and the photodetector 13, which results in an increasedsize of the apparatus. Therefore, it is preferred to use the abovedescribed approach for moving the photodetector 13. In addition, thefact was found that, when a weak light emission likebiochemiluminescence for a trace amount of ATP is measured, a simpleoperation to take out a sample container from a container holder andthen insert the container to the same holder considerably influences theresulting measurements and may cause errors. The reason of the influencehas not resolved in detail yet, but a slight change in the state of theelectrostatic charge on a sample container may adversely affect themeasurements. In spite of the reasons, the approach to move a samplecontainer and its holder has a potential to cause the problem of error.While, when the sample container 4 is fixed to prevent any change in anelectrostatic charge state and the photodetector 13 under static controlis moved, similar to the above described approach to move thephotodetector, the elimination of the potential can be effected.

The control of a distance between the sample container 4 and thephotodetector 13 can be achieved by storing a moving distance parameterof the second actuator 12 for each type of the sample container 4 in astoring medium of the control device 3 in advance so as to read out theparameter as needed. The bottom of the sample container 4 must notcontact the entrance window 16 of the photodetector 13. This is becausea high voltage is applied to the photocathode surface area of thephotodetector 13 and so the entrance window 16 is electrically chargedto some degree. Also, since the sample container 4 is often made ofplastic which is electrostatically charged, a simple access to thecontainer 4 may cause an electrostatic discharge. The distance betweenthe sample container 4 and the entrance window 16 is desirably setwithin a range from a several hundred micrometers to a severalmillimeters.

An improved sensitivity can be expected when the sample container holder5 efficiently guides the chemiluminescene in the sample container 4 tothe entrance window 16. In order to collect the chemiluminescene whichis scattered in the directions other than that toward the entrancewindow 16, specular reflection is often used. The container holder ispreferably processed with a metal material, or the inner surface of thesample container holder 5 is formed with a member having a metal film 17thereon, as shown in FIGS. 3A-C. It is advantageous, in terms of cost,to make a moldable resin material coated with a metal film. The metalmaterial is preferably silver or aluminum which provides a stablereflection efficiency of 80% or more.

FIG. 3 shows typical three types of the sample container holder 5 havinga metal film for specular reflection 17. A circular cylindrical samplecontainer holder 68 is the form which allows the most efficient approachof the photodetector 13 relative to the size of the sample container 4,but provides a low collection efficiency of reflected chemiluminescenceto the entrance window 16 of the photodetector 13. The reference numeral18 designates a tapered sample container holder, and the referencenumeral 19 designates a hemispherical sample container holder. Thesemetal films enable the guiding of the chemiluminescene scattered from aluminous point to the entrance window 16 of the photodetector 13.

Second Embodiment

FIG. 4 is a view showing a schematic structure of an apparatus forchemiluminescent assay and detection according to a second embodiment.The apparatus for chemiluminescent assay and detection of the presentembodiment includes a solution dispenser in addition to the structure ofthe first embodiment (a chemiluminescence measuring apparatus having adispenser).

In FIG. 4, a sample container 4 is mounted to a sample container holder5. The sample container holder 5 is placed over a through hole 8 whichis formed in a top plate 7 of a second light shielding BOX 6. The samplecontainer holder 5 is aligned in the same manner as that of the firstembodiment.

The top plate 7 of the second light shielding BOX 6 is configured tohave a plate member 9 inserted therethrough. The inserted plate member 9is able to move in the direction of the y-axis through the top plate 7by the first actuator 10. The through hole 8 functions as anopenable/closable window for introducing chemiluminescene by themovement of the plate member 9.

The second light shielding BOX 6 stores a photodetector 13 therein. Thephotodetector 13 is able to move in the direction of the z-axis by asecond actuator 12.

The dispenser is configured with a dispensing nozzle 20 from which asolution exits when the solution is dispensed into the sample container,a feed pump 23, a liquid supply pipe 22 which connects between thedispensing nozzle 20 and the feed pump 23, and a piping connector 21 forfixing the dispensing nozzle 20 and connecting between the dispensingnozzle 20 and the liquid supply pipe 22. The position of the dispensingnozzle 20 is controlled in the direction of the z-axis by a thirdactuator 24. The third actuator 24 may be, for example, mounted to agate-shaped plate member which is attached to the first light shieldingBOX 6.

Preferably the sample container 4, the sample container holder 5, thethrough hole 8, the photodetector 13, and the dispensing nozzle 20 arecoaxially aligned with each other at the center thereof in the directionof the z-axis.

FIG. 5 is a view showing the operation principle of the apparatus forchemiluminescent assay and detection according to the second embodiment(FIG. 4: a chemiluminescence measuring apparatus having a dispenser).FIG. 5 shows an example in which a chemiluminescence reagent, that is asubstance or enzyme, and also substance+enzyme is added through thedispensing nozzle 20 into the sample container 4 so that the sample 25in the sample container 4 having a chemiluminescent material emitsluminescence. Of course, alternatively, another form may be used inwhich a chemiluminescence reagent may be stored in the sample container4 in advance, and a sample to be measured may be supplied through thedispenser. When a trace amount of sample is measured, the latter form inwhich a sample is supplied through the dispenser is more preferable.Also, when a chemiluminescence reagent itself has some luminescencesignals, the latter form in which a sample is supplied through thedispenser is more preferable.

FIG. 5A shows a state in which the plate member 9 moves in the directionof the y-axis, and the second actuator 12 is driven, and thephotodetector 13 gets closer to the bottom of the sample container 4 inresponse to an instruction from the control device 3 to start ameasurement. FIG. 5A further shows the moment when the dispensing nozzle20 is inserted in the sample container 4. FIG. 5B shows the state that achemiluminescence reagent is dispensed from the dispensing nozzle 20,and a dispensed droplet 26 is removed from the dispensing nozzle 20,while FIG. 5C shows the state that the dispensed droplet 26 is releasedinto the sample 25 which contains a chemiluminescent material in thesample container 4 to form a luminescent solution 27.

The application of an HV to the photodetector 13 may be conducted at anytiming shown in FIGS. 5A-5C. Depending on the intention of a user, anytiming to apply an HV can be set using the control device 3.

Next, an experiments to construct a calibration curve which shows thedetection sensitivity and quantitativity of an ATP amount which isobtained using an apparatus for chemiluminescent assay and detection ofthe present embodiment, and the results of the experiment will beexplained below. A luciferase & luciferin based chemiluminescencereagent was added to ATP to obtain chemiluminescent signals.

In advance, an ATP concentrated solution having a high concentration isdiluted with a buffer such as deionized water and tris (hydroxymethyl)aminomethane solution, and the obtained ATP solution within a range from1 amol to 100000 amol is stored in the sample container 4. Achemiluminescence reagent is stored in the feed pump 23 of the dispenserwhich has a solution reservoir. As an initialization operation of theapparatus, the second actuator 12 is driven to cause the photodetector13 to be separated from the through hole 8, and the through hole 8 isclosed by the plate member 9 to block any light entry to the inside ofthe second light shielding BOX 6 (to establish the state shown in FIG.2A).

FIG. 6 is a flowchart illustrating a (typical) procedure for measuringchemiluminescence. First, the open/close door 2 of the first lightshielding BOX 1 is opened (S601), and the sample container 4 having theATP solution stored therein is placed in the BOX 1 (S602). Then, theopen/close door 2 of the first light shielding BOX 1 is closed (S603).Next, an HV is applied to PMT which is the photodetector 13 (S604). Theplate member 9 which is provided to the top plate 7 of the second lightshielding BOX 6 moves (S605), and the PMT is moved by the secondactuator 12 into the through hole 8 of the top plate 7 to get closer tothe sample container 4 which is placed at a higher position in thedirection of the z-axis than that of the plate member 9 (S606). Afterthe movement of the optical system is completed, a measurement isstarted.

The measurement is started before a chemiluminescence reagent isdispensed from the dispenser 20, and the solution signals in the samplecontainer 4 are measured as background data (S607). After themeasurement of solution signals as background data for a certain periodof time, the chemiluminescence reagent is dispensed from the dispenser20 (S608). The chemiluminescence reagent reacts with the ATP in thesample container, and a chemiluminescent reaction is started in thecontainer. After the measurement of ATP chemiluminescence for a certainperiod of time (S609), the HV to a PMT is turned off (S610), and thesecond actuator 12 is driven to move the tip portion of thephotodetector 13 to a position below the plate member 9 in the directionof the z-axis (S611). After the movement of the PMT, the plate member 9moves back to a position before the measurement, which closes thethrough hole 8 (S612). Next, in order to remove the sample container 4after measurement, the open/close door 2 of the first light shieldingBOX 1 is opened (S613), and the sample container 4 is removed (S614). Tostart measurement of a next sample, a container having the sampletherein is placed at this step, and the above described flow formeasurement is repeated. To end the measurement, after the samplecontainer 4 is removed, the open/close door 2 of the first lightshielding BOX 1 is closed to end the measurement (S615).

Desirably, among the steps shown in FIG. 6, those except the step ofopening the first light shielding BOX (S601), the step of placing thesample container (S602), the step of closing the first light shieldingBOX (S603), and the step of removing the sample container (S614) areautomatically conducted, and a user only has to press a start buttonprovided on the control device 3 for a serial execution of the steps.The waiting time for each step is not indicated herein, but may bechanged and set as needed as a parameter which can be set using thecontrol device 3.

FIG. 7A is a graph showing a typical example of a chemiluminescencecurve 44 relative to time which is obtained in the steps shown in FIG. 6using the device shown in FIG. 5. The horizontal axis represents time,while the vertical axis represents the number of photons per unit (CountPer Second: CPS). The background light signals 45 were obtained for 100seconds before a chemiluminescence reagent was dispensed, and then thechemiluminescence reagent was dispensed, and after the dispensation, ATPchemiluminescent signals 46 were obtained for 250 seconds. After thechemiluminescence reagent was dispensed, a flash type (highly sensitivetype chemiluminescence reagent is immediately added which shows a peakvalue 48 of the signal intensity in a few seconds.

FIG. 7B is a graph 47 showing a typical example of a chemiluminescencecurve, in which the differences between the peak values and the valuesof background light signals 44 of FIG. 7A are converted into numericalvalues and plotted relative to each concentration. The calibration curve49 is linear within a range from a very low concentration of 1 amol to100000 amol, which indicates a quantitative change of intensity. Thisresult is obtained because the apparatus for chemiluminescent assay anddetection of the present embodiment is highly sensitive andquantitative. In the present embodiment, any influence of entrance ofstray light onto the background light signals 45 when the samplecontainer 4 is exchanged is eliminated by the plate member 9 of thesecond light shielding BOX 6, and also the photodetector gets closer tothe container for measurement to achieve a large solid angle. Therefore,the calibration curve 47 of FIG. 7B is an ideal calibration curve as aresult obtained by measuring weak chemiluminescent signals with highsensitivity at a very low concentration.

Furthermore, the calibration curve graph 47 of FIG. 7B is important tocount the number of a trace amount of microbes, on the order of theseveral number of microbes for example. This is the indispensable datafor an experimental system for monitoring the number of airbornemicroorganisms in a clean room using the microbe count functionaccording to a third embodiment which will be explained below.

Meanwhile, when an ATP measurement at a high concentration is requiredto examine a high water contamination level and the like, thecalibration curve of FIG. 7B cannot be used to evaluate thecontamination level. However, in the apparatus for chemiluminescentassay and detection of the present embodiment, since the distancebetween the photodetector 13 and the bottom of the sample container 4can be controlled as needed, any calibration curve can be constructeddepending on a required concentration range. Specifically, when theupper limit of a dynamic range is too low for a sample to be detectedwhich has an extremely high luminescence intensity, the distance betweenthe photodetector 13 and the bottom of the sample container 4 can becorrespondingly increased. For example, when the second actuator 12which is electrically driven is used, since the moving distance isdetermined by the number of pulses sent to the motor, a distance properto each of detectable sensitivity ranges is found in advance byconducting an experiment, and the number of pulses at the experiment isstored in a storing medium of a controller. As need arises, a propernumber of pulses is selected to quickly change the distance to thecontainer. When the second actuator 12 which is driven by air is used, astopper is provided to physically control the distance. That is, aposition of the stopper depending on sensitivity is found by conductingan experiment in advance, which enables the distance control.

Third Embodiment

A third embodiment relates to an apparatus for chemiluminescent assayand detection having a microbe count function for counting the number ofliving microbes. The apparatus selectively detects only ATP which iscontained in the living microbes, and measures the amount of the ATP.The ATP content of each type of microbes is already known, thereby thenumber of microbes can be calculated based on the calibration curve ofFIG. 7B according to the second embodiment. For example, the ATP contentof one Escherichia coli is 2 amol to 3 amol.

FIGS. 8A and 8B and FIG. 9 are views showing the outline of a systemincluding an apparatus for chemiluminescent assay and detection 50having a microbe count function according to the third embodiment andthe control device 3. The apparatus for chemiluminescent assay anddetection 50 having a microbe count function is automatically controlledby the control device 3. The command from the control device 3 causesthe openable/closable window 51 to be in an open state, so that alight-shielding stage 52 is moved outside of the apparatus, on which asample preparation container 53 and a chemiluminescence detectioncontainer 55 are placed (FIGS. 8A and 8B). Single-purpose containers 53and 55 are mounted to a sample preparation container holder 54 and achemiluminescence detection container holder 69 respectively.

With reference to FIG. 9, the structure of the apparatus forchemiluminescent assay and detection 50 having a microbe count functionwill be explained below. The apparatus for chemiluminescent assay anddetection 50 includes three dispensers and one fluid dispenser, and twoof the dispensers is means for dispensing a reagent to the samplepreparation container 53, and the other one of the dispensers is meansfor dispensing a chemiluminescence reagent to the chemiluminescencedetection container 55. The fluid dispenser is means for collecting thesolution after preparation in the sample preparation container 53 anddispensing the collected solution into the chemiluminescence detectioncontainer 55. In the present embodiment, the reagent to be dispensedother than a chemiluminescence reagent is an ATP eliminating reagent andan ATP extracting reagent. The dispensing nozzles 20 are provided with agroup of third actuators 24 for the movement in the direction of thez-axis and a fifth actuator 57 for the movement in the direction ofx-axis, which allows the dispensing nozzles 20 to be inserted in thesample preparation container 53, and controls the positions of thedispensing nozzles both in the x-axis and the z-axis. The fifth actuator57 is, for example, mounted to a gate-shaped plate member which isattached to the wall of the first light shielding BOX 6, similar to thesecond actuator 24 of the first embodiment.

The light-shielding stage 52 includes the photodetector 13 therein withthe photodetector 13 being mounted to the second actuator 12. Thelight-shielding stage 52 is movable in the direction of the y-axis by afourth actuator 58. The light-shielding stage 52 has a top plate havinga through hole formed therein, over which the chemiluminescencedetection container holder 69 is placed. The apparatus forchemiluminescent assay and detection 50 further includes a tubular lightshielding attachment 56. The light shielding attachment 56 enables alight shielding, as shown in FIG. 11, when the PMT moves in thedirection of the z-axis so that the tip portion of the photodetector 13is compressed against and sealed by the light shielding attachment 56.Since the light shielding attachment 56 is made of a resilient material,a pressing of the photodetector 13 in the direction of the z-axisagainst the light shielding attachment 56 enables blocking of straylight. As the resilient material, for example, a black viton rubber/orfluorine contained rubber which is used to make O-rings for leakprevention of vacuum apparatuses is preferable. That is, in the stateshown in FIG. 8B, the through hole of the light-shielding stage 52 isnot covered with the sample preparation container holder 54 or thechemiluminescence detection container holder 69, the protection of asensitive area of the photodetector 13 is needed. Thus, in the stateshown in FIG. 8B, the tip portion of the photodetector 13 is pressedagainst the light shielding attachment 56, so that the sensitive area ofthe photodetector 13 is protected from stray light.

Next, with reference to FIG. 10, a procedure for counting livingmicrobes using the apparatus shown in FIG. 9 will be explained below.First, an openable/closable window 51 is opened (S1001), and thelight-shielding stage moves (S1002). At this step, the samplepreparation container 53 and the chemiluminescence detection container55 in which a cell suspension containing collected microbes therein arestored are placed (S1003). After the placement, the light-shieldingstage is moved into the microbe count apparatus 50 (S1004). Then, theopenable/closable window 51 is closed (S1005).

Next, an ATP eliminating reagent is dispensed in the sample preparationcontainer 53 to eliminate the exogenous ATP except living microbe andthe ATP from killed microbe (S1006). After the reaction is completed, anATP extracting reagent is dispensed in the sample preparation container53 (S1007), and the light-shielding stage 52 is moved to a positionwhich allows a dispensation from the chemiluminescence detectioncontainer 55 (S1008). Next, a chemiluminescence reagent is dispensed inthe chemiluminescence detection container 55 (S1009). However, thedispensation of the chemiluminescence reagent may be conductedbefore/after the injection of the ATP eliminating reagent.

The sample solution which is already prepared to be dispensed in thechemiluminescence detection container 55 is collected from the samplepreparation container 53 before the measurement is started (S1010). Theamount of the collected solution should be adjustable within a rangefrom several microliters to several milliliters, and the system of thefeed pump 23 preferably uses a combination of a syringe and a syringepump. In the above steps, the state shown in FIG. 11 is a characteristicwhich constrains light accumulation due to stray light when a containeris placed.

Then, the photodetector 13 moves in the direction of the z-axis (S1011)to be inserted in the through hole of the top plate, and an HV isapplied to PMT which is the photodetector 13 (S1012). After backgroundlight signals are measured from under the bottom of thechemiluminescence detection container to which the chemiluminescencereagent was dispensed (S1013), the collected sample solution afterpreparation is dispensed in the chemiluminescence detection container 55(S1014). The ATP contained in the prepared sample solution reacts withthe chemiluminescence reagent and emits chemiluminescence. The ATPchemiluminescence is measured (S1015). Then the HV applied to PMT isturned off (S1016), and the second actuator 12 is driven to cause thetip portion of the photodetector 13 to move in the direction of thez-axis to a position below the top plate 7 of the light-shielding stage52, and also move in the direction of the y-axis, so that the tipportion of the photodetector 13 is compressed and sealed by the lightshielding attachment 56 (S1017).

The resulting ATP luminescence intensity and the calibration curve ofFIG. 7B according to the second embodiment are used to calculate thenumber of microbes, and the data is sent (S1018). In other words, thenumber of microbes can be calculated based on the known ATP amount ofeach microbe. For example, bacillus subtilis contains 17 amol/cell,staphylococcus aureus contains 1.52 amol/cell, and Escherichia colicontains 3 amol/cell.

In the present embodiment, a chemiluminescence measuring apparatushaving a microbe count function for counting the number of livingmicrobes which uses the biology chemiluminescence reaction of ATP, butthe applicable scope of an automation equipment which includes anautomatic specimen preparation mechanism for automatic samplepreparation and a chemiluminescence detection mechanism with highsensitivity is not limited to the microbe count function. As a modifiedexample of the present embodiment, for example, a system forautomatically measuring an amount of chemiluminescence can be achievedin which a specimen solution containing an antigen of a concentrationwhich is not known is used as a specimen, and a chemiluminescencereagent containing an excess amount of luciferin and ATP is used as achemiluminescence reagent, and a sample preparation mechanism is used,so that an antigen-antibody reaction in so-called sandwich immunoassaytechnique is initiated with respect to the specimen antigen to produce areaction solution which contains a luciferase labeled products of theamount which is proportional to the amount of the antigen, and then thereaction mixture is dispensed through a fluid dispensing mechanism intothe chemiluminescence detection container 55 which stores achemiluminescence reagent therein. The amount of chemiluminescence inthe modified example is proportional to the amount of luciferase in thereaction mixture, that is the amount of an antigen-antibody reaction.Therefore, an immunoassay system with high sensitivity for measuring theamount of antigen by comparing with the amount of chemiluminescence of astandard antigen specimen having a known concentration can be achieved.The system can be applied to an apparatus for assay and detection of DNAand RNA with high sensitivity in which nucleic acid hybridization isused as a selective combination principle and luciferase is a label.

SUMMARY OF EMBODIMENTS

The first embodiment provides a chemiluminescence measuring apparatus ofdouble light shielding type which includes a first light shielding BOXhaving an open/close door that is used to place or remove a samplecontainer from a sample container holder, and a second light shieldingBOX in the first light shielding BOX, the second light shielding BOXhaving a top plate which is partly configured as a shutter unit and hasan openable/closable mechanism to see the first light shielding BOXtherethrough, and has a photodetector housed therein. This completelyblocks stray light to carry out a measurement of chemiluminescence at ahigh sensitivity and accuracy.

As to the specific structure for light shielding, basically, the topplate of the second light shielding BOX has at least one through holeformed therein for opening and closing, and a container holder in whicha sample container is placed is placed over the through hole, and theshutter functions to open and close the through hole. A photodetector isplaced on the bottom via an electrically operated actuator, so that whenthe shutter of the top plate of the second light shielding BOX isopened, a sensitive area of the photodetector in the second lightshielding BOX is positioned opposite to the bottom of the samplecontainer which is placed in the sample container holder.

When the first light shielding BOX is opened to place the containerwhich stores a chemiluminescent material therein, the shutter of the topplate of the second light shielding BOX is closed. This blocks theentrance of stray light to the photodetector which may cause a variationin dark current values. After the container which stores achemiluminescent material therein is placed, the first light shieldingBOX is closed. When a measurement is started, the shutter at the topplate of the second light shielding BOX is opened, and a sensitive areaof the photodetector is inserted into the through hole by theelectrically driven actuator. The electrically driven actuator cancontrol the distance between the bottom of the sample container and thesensitive area of the photodetector as needed. The sensitive area of thephotodetector may be positioned above the position of the shutter fordouble light shielding, which achieves a close arrangement to the bottomof the sample container. In this state, in order to measurechemiluminescence, the chemiluminescence can be efficiently collected tothe sensitive area, and a so-called large solid angle can be formed,thereby chemiluminescence can be detected with high sensitivity andaccuracy.

The second Embodiment is provided with means (such as a nozzle, a feedpump, and a liquid supply pipe) for dispensing a solution to a samplecontainer in addition to the structure of the first embodiment.

Furthermore, the third embodiment provides an apparatus forchemiluminescent assay and detection with a microbe count function. Themicrobe count function can be achieved by providing a group of nozzlesfor collecting/dispensing a treatment solution for a reaction which isrequired to measure the amount of ATP from living microbes, solutionsupply pipes which are connected to the nozzles, a solution storingcontainer for storing the treatment solution, and a pump which is themeans for collecting/dispensing the solution through the tips of thenozzles, in the first light shielding BOX of the above describedapparatus for chemiluminescent assay and detection of double lightshielding type. The container holder for holding a container formeasuring chemiluminescence is placed over the through hole formed inthe top plate on the second light shielding BOX, and at least oneanother container holder which can have a container placed therein isplaced at another position. Hereinafter, the latter container which isused to prepare a sample is referred to as a sample preparationcontainer.

An ATP eliminating reagent is dispensed from the nozzle into the cellsuspension in the specimen preparation container to eliminate killedmicrobes and floating ATP other than living microbes. Next, an ATPextracting reagent is dispensed to extract ATP of living microbes. Inthe above process, the photodetector is shielded from light by thesecond light shielding BOX. Next, a chemiluminescence reagent isdispensed in the container for measuring chemiluminescence, and finallya treated cell suspension is collected to be dispensed and mixed in thechemiluminescence reagent. Upon the mixture, or just before the mixture,a sensitive area of the photodetector is moved to a position above thetop plate (the through hole is formed therein) of the second lightshielding BOX so that the photodetector starts a measurement at aposition closer to the container for measuring chemiluminescence. Sincebackground light data can be obtained at the time of or before thedispensation, when a so-called flash type chemiluminescence reagent isused, the signal of the highest chemiluminescence just after thereaction is started can be obtained.

It is possible to add the structure of the third embodiment to that ofthe first or second embodiment. For example, the light shieldingattachment 56 (see FIG. 11) may be added to the structure of the firstor second embodiment. This guarantees a better light shielding property.

1. An apparatus for chemiluminescent assay and detection, comprising: acontainer for storing a specimen; a holder for holding the container; aphotodetector which is provided opposite to the container; a platemember which is provided opposite to the photodetector; a plate memberdriving section for moving the plate member relative to thephotodetector; and a photodetector position control section for movingthe photodetector relative to the container, wherein the photodetectoris provided opposite to the container via the plate member, and thephotodetector position control section moves the photodetector so that,when at least a part of the plate member is moved, an end surface of thephotodetector is placed at the same position as that of a surface of theplate member opposite to the photodetector or at a position closer tothe container than the opposite surface.
 2. The apparatus forchemiluminescent assay and detection according to claim 1, wherein theplate member has at least one through hole formed therein.
 3. Theapparatus for chemiluminescent assay and detection according to claim 1,wherein the plate member has at least one through hole formed therein,and at least a tip portion of the detector which is opposite to theplate member is inserted into the through hole.
 4. The apparatus forchemiluminescent assay and detection according to claim 1, wherein theplate member has at least one through hole formed therein, and at leasta tip portion of the detector which is opposite to the plate memberpasses through the through hole to be positioned in the holder.
 5. Theapparatus for chemiluminescent assay and detection according to claim 1,wherein the plate member moves in a direction perpendicular to thedirection in which the photodetector moves.
 6. The apparatus forchemiluminescent assay and detection according to claim 1, wherein thephotodetector position control section controls the distance between abottom of the container and the photodetector which is opposite to thecontainer in response to the intensity of chemiluminescence signals. 7.The apparatus for chemiluminescent assay and detection according toclaim 1, wherein the holder for holding the container with the specimenstored therein has an inner wall which opposes to the container and iscovered with a metal film.
 8. The apparatus for chemiluminescent assayand detection according to claim 1, wherein a part of the inner wall ofthe holder for holding the container has a tapered shape.
 9. Theapparatus for chemiluminescent assay and detection according to claim 1,wherein a part of the inner wall of the holder for holding the containerhas a hemispherical shape.
 10. An apparatus for chemiluminescent assayand detection, comprising: a container for storing a specimen; a holderfor holding the container; a photodetector which detects luminescencefrom the container; a plate member which is provided between thephotodetector and the holder; a plate member driving section that movesthe plate member from between the photodetector and the holder; and aphotodetector position control section for moving the photodetectorrelative to the container, wherein the photodetector position controlsection moves the photodetector so that an end surface of thephotodetector is placed at the same position as that of a surface of theplate member opposite to the photodetector or at a position closer tothe container than the opposite surface.
 11. The apparatus forchemiluminescent assay and detection according to claim 10, furthercomprising a holding member of the plate member, wherein the holdingmember of the plate member has at least one through hole formed therein.12. The apparatus for chemiluminescent assay and detection according toclaim 10, further comprising a holding member of the plate member,wherein the holding member of the plate member has at least one throughhole formed therein, and at least a tip portion of the detector which isopposite to the plate member is inserted into the through hole.
 13. Theapparatus for chemiluminescent assay and detection according to claim10, further comprising a holding member of the plate member, wherein theholding member of the plate member has at least one through hole formedtherein, and at least a tip portion of the detector which is opposite tothe plate member passes through the through hole to be positioned in theholder.
 14. The apparatus for chemiluminescent assay and detectionaccording to claim 10, wherein the plate member moves in a directionperpendicular to the direction in which the photodetector moves.
 15. Theapparatus for chemiluminescent assay and detection according to claim10, wherein the photodetector position control section controls thedistance between a bottom of the container and the photodetector whichis opposite to the container in response to the intensity ofchemiluminescence signals.
 16. The apparatus for chemiluminescent assayand detection according to claim 10, wherein the holder for holding thecontainer with the specimen stored therein has an inner wall whichopposes to the container and is covered with a metal film.
 17. Theapparatus for chemiluminescent assay and detection according to claim10, wherein a part of the inner wall of the holder for holding thecontainer has a tapered shape.
 18. The apparatus for chemiluminescentassay and detection according to claim 10, wherein a part of the innerwall of the holder for holding the container has a hemispherical shape.